JP2023504773A - AAV vector variants for ocular gene delivery - Google Patents

Abstract

The present invention relates to adeno-associated virus capsid polypeptide sequences and their use in therapeutic transgene delivery to the eye and potentially other tissues. [Selection figure] None

Description

The present invention relates to adeno-associated virus capsid polypeptide sequences and their use in therapeutic transgene delivery to the eye that targets photoreceptors or retinal pigment epithelial cells.

Permanent degeneration of light-sensitive retinal photoreceptor cells (PR) causes blindness. Death of photoreceptors can be caused by inherited mutations that are primarily localized to PR or retinal pigment epithelial (RPE) cells, such as in retinitis pigmentosa (RP), or, for example, in age-related macular degeneration (AMD). It can be triggered by multifactorial conditions that affect cellular health. Genetic mutations responsible for many forms of photoreceptor degeneration have been identified, allowing intervention by administration of therapeutic transgenes. Current PR-targeted nucleic acid-based therapies are effective only at early disease stages where PR is minimally damaged. Gene therapy with a suitable visual gene protein that confers light sensitivity to functional retinal lining cells may potentially restore vision even at later stages of the disease. To make such an approach practical in the medical setting, improved vectors with better retinal penetration for gene delivery are needed.

Adeno-associated virus (AAV)-based recombinant vectors are candidates for therapeutic gene transfer in the eye. Currently, AAV vectors are typically administered subretinally. However, subretinal medication has been shown to result in decreased retinal thickness and visual acuity. In addition, subretinal administration effects gene transfer and expression only in cells directly adjacent to the injectate mass, whereas an effective administration method should reach cells along the entire width of the retina. Intravitreal injection into the jelly-like filling of the eye (vitreous humor) not only has the potential to deliver vectors throughout the retina, but is also significantly safer and technically more demanding than subretinal injection. is much lower. However, the inner limiting membrane (ILM), which separates the neural retina from the vitreous humor, is enriched with natural receptors for many AAV serotypes, thereby building a strong barrier against AAV vectors. Engineered AAV vectors that penetrate better through the ILM and other retinal barriers when delivered intravitreally are needed to increase the efficacy and safety of ocular AAV gene therapy.

Based on the above state of the art, the object of the present invention is for gene transfer into the retina, which makes it possible to target all retinal cell types, in particular retinal inner layer cells and photoreceptors. is to provide a means and method of This object is achieved by the subject matter of the independent claims herein.

A first aspect of the invention is an adeno-associated virus (AAV) capsid polypeptide, which is located in the highest and second highest capsid overhangs, respectively, of the AAV serotype 2 capsid at positions 453 or 587/588 or Adeno-associated virus (AAV) capsid polypeptides containing peptide insertions at positions homologous to those in AAV capsids of other serotypes (Table 3: #1, #2). The peptide insert has the following sequence:
SASEAST (Cap3; SEQ ID NO:10), DTRPHDQ (Cap5; SEQ ID NO:11), EHYNSTC (Cap7; SEQ ID NO:12), PNPNCTL (Cap9; SEQ ID NO:13), TPPSITA (Cap11; SEQ ID NO:14), CGESSYL (Cap12; number 15), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17)
is selected from

The peptide may be flanked by short stretches of other amino acids (1, 2, 3, 4, 5 or even 6AA). Particular examples of flanking amino acids to fill in the indicated sequence at the indicated position within the capsid sequence can be selected from (but are not limited to) Ala, Leu, Gly, Ser and Thr.

The peptide inserts increase the efficiency of adeno-associated virus infection and/or the transduction efficiency of adeno-associated vectors representing peptide inserts on the capsid surface at the positions indicated above in I-587, at least for the cell types and tissues tested. increase.

Peptide inserts may act similarly with I-588 or I-453 due to their cell surface exposure. Without wishing to be bound by theory, the inventors believe that at least part of the effect is due to lower binding to heparan sulfate proteoglycans in vivo.

A second aspect of the invention relates to a nucleic acid sequence encoding an AAV capsid polypeptide according to the first aspect. Particular embodiments include inclusion of this nucleic acid sequence in the AAV cap sequence, particularly the AAV serotype 2 capsid sequence, for the production of capsid engineered AAV vectors.

A third aspect of the invention relates to agents selected from AAV capsid polypeptides, AAV vectors and nucleic acid sequences for the treatment of conditions affecting retina or RPE cells. An alternative form of this aspect is realized by a method for the treatment of a condition affecting photoreceptors or RPE cells comprising administering an agent according to the invention to a patient in need thereof.

Dosage forms containing agents of the invention are a further aspect of the invention.

Retinal layer specificity of expression for selected capsid variants. Example of pan-retinal transgene expression throughout the outer mouse retina after intravitreal injection. Comparison of novel capsid-delivered transgene expression with AAV2(M6) and AAV2(7m8) after intravitreal injection into mouse eyes.

Terms and Definitions The abbreviation AAV as used herein relates to adeno-associated virus.

The term AAV vector in relation to this specification relates to a viral vector composed of 60 AAV capsid proteins and encapsidated AAV nucleic acid. AAV vectors are derived from AAV virions, but AAV vectors have been engineered to be unable to replicate in the presence of a helper virus by removing the rep and cap genes from the AAV genome. Encapsidated AAV nucleic acids may contain transgenes that are delivered to target cells.

Unless otherwise defined, all technical and scientific terms used herein are commonly understood by those of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). have the same meaning. Standard techniques include molecular, genetic and biochemical methods (generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term AAV capsid as used herein relates to a polypeptide encoded by an engineered capsid (cap) gene produced by the inventors and listed herein below. The AAV capsids disclosed herein can be used to assemble recombinant adeno-associated virus vectors for gene therapy.

References to amino acid positions in the AAV capsid herein refer to the capsid amino acid sequence of adeno-associated virus 2 capsid protein VP1 having the sequence of SEQ ID NO: 1 (Table 2) (the GenBank accession number for the corresponding nucleic acid sequence is J01901.1). Corresponding amino acid positions for other homologous adeno-associated virus serotypes are shown in Table 3.

The term homologous as used herein relates to nucleic acid or amino acid sequences derived from different serotypes of AAV. The corresponding positions of various adeno-associated virus serotypes are shown in Table 3.

The term transgene as used herein relates to a gene or genetic material transferred from one organism to another. In the context of this specification, the term can also refer to the transfer of a natural or physiologically intact variant of a gene sequence into tissues of a patient in which it has been deleted. The term may further refer to the movement of the native coding sequence, expression driven by a promoter that is absent or silenced in the target tissue.

The term recombinant in the context of this specification relates to nucleic acids which are the product of one or several steps of cloning, restriction and/or ligation and differ from naturally occurring nucleic acids. A recombinant viral particle contains a recombinant nucleic acid.

The term intravitreal administration in relation to this specification relates to a route of administration of a pharmaceutical agent, eg a viral vector, wherein the agent is delivered into the vitreous of the eye. Intravitreal administration is the procedure of placing medications directly into the posterior space of the eye, called the vitreous cavity, which is filled with a jelly-like fluid called vitreous gel.

The term subretinal administration as used herein relates to the route of administration of a pharmaceutical agent, in particular a viral vector as used herein, into the space between the cells of the retinal pigment epithelium (RPE) and the photoreceptors.

The term polypeptide as used herein relates to molecules consisting of 50 or more amino acids forming a linear chain, the amino acids being linked by peptide bonds. The amino acid sequence of a polypeptide can refer to the entire (as found physiologically) protein amino acid sequence or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. A polypeptide is disclosed herein as a sequence of amino acid residues.

Sequences of amino acid residues are given from the amino terminus to the carboxyl terminus. Capital letters at sequence positions refer to L-amino acids in the one-letter code ( ^Stryer , Biochemistry, 3rd ed. p. 21). Lowercase letters at amino acid sequence positions refer to the corresponding D- or (2R)-amino acid. Sequences are written left to right in the direction from amino-terminus to carboxy-terminus. In accordance with standard nomenclature, amino acid residue sequences are designated by three-letter or single-letter codes as shown below: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T ), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

The term variant refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in its primary amino acid sequence from another polypeptide used as a reference. In general, differences are limited and the sequences of the reference polypeptide and variant are generally closely similar and identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (eg, substitutions, additions and/or deletions). Variants of a polypeptide may be naturally occurring, such as allelic variants, or may be variants not known to occur naturally.

The term biological activity in relation to this specification relates to a finely measurable quantity exhibited by certain variants of the polypeptides included herein. For example, with respect to the ability of viral vectors to facilitate gene transfer into retinal cells, the biological activity of capsid mutants can be measured in standard assays, in vivo, in transgenes into cultured cells or into model organisms (e.g. , firefly luciferase).

As used herein, the terms sequence identity and percentage of sequence identity refer to values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well known in the art. Sequence alignments for comparison are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the local homology algorithm, Needleman and Wunsch, J. Am. Mol. Biol. 48:443 (1970), Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computer implementation of these algorithms including, but not limited to, CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyzes is publicly available, eg, through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparing amino acid sequences is the BLASTP algorithm using the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; , Extension 1; Compositional adjustments: Conditional compositional score matrix adjustments. One such example for comparison of nucleic acid sequences is the BLASTN algorithm using the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; . -2; Gap costs: Linear. Unless otherwise specified, the sequence identity values provided herein are the values obtained using the BLAST suite of programs, using the default parameters specified above for protein and nucleic acid comparisons, respectively. (Altschul et al., J. Mol. Biol. 215:403-410 (1990)).

As used herein, the term amino acid linker refers to oligopeptides of variable length that are used to join two polypeptides together to produce a single polypeptide chain. An exemplary embodiment of a linker useful in practicing the invention specified herein is an oligopeptide chain consisting of 1-6 amino acids. Non-limiting examples of amino acid linkers are AAA or AA as exemplified below.

AAV is a small, nonpathogenic virus that infects humans and other primate species.

AAV2 infection is initiated by docking to the cell surface receptor heparan sulfate proteoglycan (HSPG). Its low binding affinity for glycans induces a reversible structural rearrangement of the capsid that facilitates binding to co-receptors αvβ5 or α5β1 integrins, triggering the formation of clathrin-coated pits. Clathrin-coated pits are internalized via endocytosis and virus particles are transported to the nucleus. The pH is lowered by acidification of the endosomal compartment, which is characteristic of endosomal vesicle maturation. A conformational change caused by acidification occurs in the capsid and the virus escapes from the late endosome by lipolytic pore formation.

In wild-type AAV, the genome consists of a 4.7 kilobase long single-stranded DNA (ssDNA) of either positive or negative sense. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs). ITRs are self-complementary, CG-rich, T-shaped hairpins at the 5' and 3' ends of the AAV genome and are the only necessary viral components present in the recombinant vector genome. The ITR contains a terminal breaking site (TRS) and a Rep binding element (RBE), which facilitate replication and encapsidation of the viral genome. The ORFs encode the genes rep, cap, AAP. Four multifunctional non-structural Rep proteins encoded by rep are required for the AAV life cycle. Cap is required to stabilize and transport capsid proteins VP1, VP2 and VP3, which interact together to form capsids of icosahedral symmetry, and newly produced VP proteins from the cytoplasm to the nucleus. It encodes an assembly activation protein (AAP). All three VPs are translated from one mRNA, but are spliced differently. The largest 90 kDa VP1 is an unspliced transcript, the 72 kDa VP2 is translated from the uncommon ACG start codon, whereas the smallest 60 kDa VP3 is translated from the AUG codon. . All three VPs have overlapping C-termini.

VP3 constitutes 90% of the capsid, and VP1 and VP2 share a common C-terminal amino acid sequence with VP3, but have N-terminal extensions buried inside the capsid. The unique N-terminal sequence of VP1 contains the phospholipase A2 (PLA2) activity and nuclear localization signal required for infection. The three VP monomers assemble with 2-fold, 3-fold and 5-fold axisymmetry to create an AAV capsid of 60 subunits. A first aspect of the invention relates to an adeno-associated virus (AAV) capsid polypeptide comprising a peptide insert in the peak or spike overhang at positions 453 or 587 of the AAV serotype 2 capsid. The peptide insert has the following sequence:
SASEAST (Cap3; SEQ ID NO:10), DTRPHDQ (Cap5; SEQ ID NO:11), EHYNSTC (Cap7; SEQ ID NO:12), PNPNCTL (Cap9; SEQ ID NO:13), TPPSITA (Cap11; SEQ ID NO:14), CGESSYL (Cap12; number 15), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17)
in particular the insert is selected from SASEAST (Cap3; SEQ ID NO:10), TPPSITA (Cap11; SEQ ID NO:14), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17) be.

The peptide inserts described above are selected from Ala, Leu, Gly, Ser and Thr, contained in the 7-13mer and inserted at the N- and/or C-terminus of the insert sequence shown in the previous paragraph. flanked by, but not limited to, 0-6 linker amino acids (6 being the maximum number of total number of amino acids flanked at the N- and C-termini). .

Any positions given for the AAV2 sequences shown herein are relative to the reference sequence available at GenBank entry number J01901.1 (Adeno-associated virus 2, complete genome). The corresponding amino acid sequence is shown as SEQ ID NO: 1 in Table 2.

The spike-like projections (peaks) correspond to the most exposed regions of the capsid. The highest peak is located at AAV2 capsid amino acid position 453 and the second highest peak is located at position 587. These peaks accept peptide insertions without interfering with capsid assembly and offer the opportunity to target non-permissive cells. Similarly, overhangs represent important sites for AAV host interaction, receptor binding and immunogenicity. Of note, in certain embodiments, R585 and 588 are mutated to obtain optimal potency when the insert is inserted at position 453.

When referring to the position of a peak or spike overhang into which a peptide insert is introduced, the peptide insert may be immediately (toward the C-terminus) or further 1-6 amino acids after this position (toward the C-terminus). It should be understood to obtain For example, a peptide insertion defined to be at position 587 (insertion defined as between pre-insertion positions 587 and 588) can be modified at positions 588 or 589 (with 1 or 2 more amino acids towards the C-terminus) or at position 590 (three more amino acids in the C-terminal direction). All of these insertion positions are intended for peptide insertions that do not interfere with the overall capsid structure, but rather may increase transduction efficiency.

This peptide insert increases viral retinal penetration and transduction efficiency after intravitreal delivery.

In certain embodiments, the peptide insert is SASEAST (Cap3; SEQ ID NO:10), DTRPHDQ (Cap5; SEQ ID NO:11), EHYNSTC (Cap7; SEQ ID NO:12), PNPNCTL (Cap9; SEQ ID NO:13), TPPSITA (Cap11; SEQ ID NO:14), CGESSYL (Cap12; SEQ ID NO:15), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17).

In certain embodiments, the peptide insert is selected from SASEAST (Cap3; SEQ ID NO:10), TPPSITA (Cap11; SEQ ID NO:14), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17).

Peptide insertions can be present in the AAV capsid polypeptide either in situ or with 1, 2, 3 or 4 flanking spacer amino acids at the amino and/or carboxyl terminus. Suitable spacer AA include but are not limited to alanine, leucine, glycine, threonine and serine.

In our example, due to the cloning strategy used, the insertion between N ₅₈₇ and R ₅₈₈ of AAV2 VP1 was of the form R ₅₈₅ GNAAAX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ - 12mer of AAR ₅₈₈ QAA, X1-X7 indicate inserted heptamer oligos, A indicates flanking linkers. It should be understood that this is only an example and that the invention is not limited to inserts at this exact position with sequences flanking these exact positions.

In the embodiment for the insertion position designated 587 in AAV2, the oligo is always inserted between positions 587 and 588 of VP1 of AAV2. It is an insertion, so VP1 AAV2 numbering is unchanged and subsequent positions are counted 'without insertion' for substitutions and the like. The inserted peptide contains a heptamer, as specified herein, but can also be potentially functionally shorter or longer. In the embodiment shown in the Examples, we inserted a 12mer of the form AAA-X1X2X3X4X5X7-AA, but the linker amino acids can be freely selected from Ala, Leu, Gly, Ser, Thr . N-terminal and C-terminal linkers can be 0-5 AA long. In certain embodiments, the inserted peptide can be 5-13 amino acids long, including 0-6 linker amino acids.

In certain embodiments, peptide insertions (referred to elsewhere herein as "oligos") can be moved 1-5 AA further to the C-terminus. For AAV2, the insertion site can also be amino acid 453 (eg, immediately C-terminal thereof).

According to an alternative form of this first aspect of the invention, any of the peptide sequences detailed above are inserted at position 453 of SEQ ID NO:001 in place of position 587. Furthermore, the position of the insertion may vary and the peptide sequence may consist of 5-13 AA flanked by the inserts shown above. In addition to the peptide insertion at position 453, R587A and R588A mutations can be introduced into VP1 of AAV2 (Boucas Jet al. J Gene Med. 2009 Dec;11(12):1103-13.doi:10.1002/ Jgm.1392).

According to another alternative form of this aspect of the invention, the position homologous to positions 587/588 or 453 in AAV serotype 2 is a different peptide having one of the inserted peptide sequences detailed above. (Table 3: #1, #2).

In certain embodiments, the AAV capsid protein is characterized by a tyrosine to phenylalanine substitution of one or several of the tyrosine residues, wherein the tyrosine residues are 252, 272, 444, 500, 700 in the wild-type capsid sequence. , 704 and 730.

Some of the capsid variants herein were selected by evolutionary methods for tyrosine modified capsids.

In certain embodiments, the AAV capsid protein is characterized by one or several tyrosine to phenylalanine substitutions at positions 252, 272, 444, 500, 704 and 730.

In certain more specific embodiments, the AAV capsid protein is characterized by several tyrosine to phenylalanine substitutions at all of positions 252, 272, 444, 500, 700 and 730.

According to another alternative form of this aspect of the invention, the tyrosine position homologous to any of positions 252, 272, 444, 500, 700, 704 and 730 in AAV serotype 2 is replaced by a tyrosine to phenylalanine substitution can be selected for substitutions to phenylalanine (Table 3: #3-#9) in order to construct another serotype of AAV with In addition, other tyrosine positions can be substituted with phenylalanine.

In certain embodiments, the AAV capsid protein is characterized by one or several threonine to valine substitutions, particularly T491V.

According to another alternative form of this aspect of the invention, the threonine position homologous to position 491 in AAV serotype 2 is replaced with valine to construct another serotype of AAV with a threonine to valine substitution. (Table 3: #10).

In certain embodiments, the tyrosine to phenylalanine substitution mentioned above is combined with a threonine to valine substitution, specifically T491V.

The YF and TV mutations reduce ubiquitination and thus proteasomal degradation of viral particles once internalized by the cell, thereby increasing transduction efficiency.

In certain embodiments, the adeno-associated virus capsid polypeptide comprises an amino acid sequence selected from SEQ ID NOs:2-9, or at least 85% identity thereto and a sequence selected from SEQ ID NOs:2-9. Includes sequences that have at least 90% of their biological activity.

Biological activity assay:
Whenever a polypeptide that "has at least a percentage of the biological activity of a (reference) sequence" is referred to herein, this biological activity can be measured as follows.

HEK293 cells are subjected to calcium phosphate precipitation with plasmids encoding test or reference polypeptides, plasmids carrying helper adenoviral genes and transgene plasmids selected from scCMV-mCitrine, scEf1a-mCitrine, scCMV-EGFP, scEf1a-EGFP as reporters. are co-transfected using the method. The transgene-containing vector is concentrated by density purification on an iodixanol gradient (Axis-Shield, Oslo) and the 40% iodixanol fraction is then buffer exchanged, eg by Amicon filtration (Millipore). AAV fractions are titered against DNase-resistant vector genomes by real-time PCR and compared to standard vectors. 3 μl of viral vector is injected intravitreally into anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine) wild-type C57BL/6J mice. For injection, the virus is titer equivalent to E11 GC/ml and 3 ul of it is injected, ie 3×E8 vg per eye. Mouse retinas are removed 3 weeks after injection for immunohistochemical analysis. Mouse eye retinal tissue strips (7 days post-transduction) were fixed with 4% (wt/vol) paraformaldehyde in PBS for 40 min at room temperature (RT) and treated with graded sucrose solutions (10%, 20% in PBS). % and 30% sucrose) at 4° C. for 3 consecutive nights; C. T. Embed in cryomold by compound (Sakura Finetek) and freeze in liquid nitrogen-cooled 2-methylbutane. 10-μm-thick vertical sections were cut with a cryostat, mounted on SuperFrost glass slides (Menzel), and subjected to suitable primary and retinal cell types to detect EGFP, YFP, mCitrine or Turbo635 fluorescence and specific retinal cell types. Immunohistochemical labeling with secondary antibody. Reporter expression in the target cell type is typically quantified by fluorescence and compared to the amount of reporter in the rest of the tissue to assess the biological activity of the engineered viral capsid. Another criterion for the biological activity of engineered viral capsids is the percentage of target cells transduced and the area of expressing tissue. For the avoidance of doubt, assays that quantify reporter expression in the target cell type and compare it to the amount of reporter in the rest of the tissue are used when measuring biological activity.

A second aspect of the invention relates to a nucleic acid sequence encoding an AAV capsid polypeptide according to the first aspect.

In certain embodiments, AAV sequences according to the invention do not contain Rep elements required for integration into the host genome. Non-integrating viruses are safer for administration in humans. In certain embodiments, the nucleic acid sequences are designed according to the self-complementary AAV vector genome concept. In all cases single-stranded DNA sequences are used.

In some embodiments, the nucleic acid sequence comprises a transgene with or without regulatory sequences.

In certain embodiments, the transgene is designed to target regions of mRNA for degradation of the appropriate protein (i.e., RPE65), siRNA or shRNA (wild-type and mutant RNAs of potential virulence genes). or the sequence of the CRISPR/Cas-gRNA cassette for gene editing.

In certain embodiments, the transgene encodes a light-sensitive protein, such as an invertebrate or vertebrate opsin or variant thereof.

In certain embodiments, the transgene encodes channelrhodopsin-2 or a variant thereof.

In certain embodiments, the transgene encodes a microbial photogated inhibitory ion pump such as, for example, halorhodopsin (eNpHR) or artirhodopsin (ArchT).

In certain embodiments, the transgene encodes a GPCR opsin, such as, for example, human rhodopsin, melanopsin, or cone opsin.

In certain embodiments, the transgene encodes a receptor protein for the photoswitchable ligand.

In certain embodiments, the transgene is under control of a promoter sequence operable in mammalian cells. In certain embodiments, the promoter sequence is operable in human retinal cells.

In one embodiment, the promoter is a ubiquitous promoter. In certain embodiments, the promoter is a cell-specific promoter.

In some embodiments, the promoter is the CMV immediate early promoter. In some embodiments, the promoter is the human Ef1a promoter. In certain embodiments, the promoter is a photoreceptor-specific promoter.

A third aspect of the invention relates to an AAV capsid polypeptide according to the first aspect, an AAV vector comprising the capsid polypeptide according to the first aspect and a nucleic acid sequence according to the second aspect for use in medicine/as a medicament. Regarding selected agents.

In certain embodiments, AAV vectors are used to transduce ocular cells.

The eye is made up of three layers composed of different anatomical structures. The fibrous membrane is the outermost layer and is composed of the cornea and sclera. The vascular membrane or uvea is the middle layer and consists of the choroid, ciliary body, pigment epithelium and iris. The retina is the innermost layer and receives oxygen from the choroidal (posterior) and retinal (anterior) vessels.

The space between the cornea and the lens is filled with aqueous humor, and the entire posterior cavity behind the lens is filled with a jelly-like substance, the vitreous. The vitreous is composed of water, collagen, fibrils, hyaluronic acid and ions. Spaces in the retina that are occupied by neither neurons nor blood vessels are filled by processes of Müller glial cells that span all retinal cell layers.

The outer limiting membrane (OLM) of the retina is formed from the junctions between Müller cells (MCs) and the inner segment of photoreceptor cells and acts as a metabolic barrier between the subretinal spaces, allowing the passage of large molecules. is restricted. The inner limiting membrane (ILM) of the retina is formed by the adjacent junctions between the MC endfeet and the basement membrane and acts as a diffusion barrier between the vitreous humor and the neural retina.

The retina is composed of the macula, optic disc, fovea fovea and peripheral retina. Photoreceptor cells are a special type of neuroepithelial cell found in the retina. Three types of photoreceptor cells are known: rods, cones and photosensitive retinal ganglion cells. Rods are distributed in the peripheral regions of the retina, whereas the central pigmented region called the macula is rich in cone photoreceptor cells. The retinal pigment epithelium (RPE) provides nutrition and maintains the health of photoreceptor cells. The nuclei of photoreceptor cells make up the outer nuclear layer (ONL), whereas the nuclei of bipolar, amacrine and horizontal cells are located in the inner nuclear layer (INL).

In certain embodiments, an agent selected from an AAV capsid polypeptide according to the first aspect, an AAV vector comprising a capsid polypeptide according to the first aspect and a nucleic acid sequence according to the second aspect comprises
a. retinal cells or retinal pigment epithelial cells, and/or b. For use in the treatment of conditions affecting photoreceptor, bipolar, amacrine or ganglion cells of the retina.

In certain embodiments, the agent is glaucoma, retinitis pigmentosa, macular degeneration, retinal detachment, Leber congenital amaurosis, diabetic retinopathy, color blindness or color blindness, melanoma-related retinopathy, congenital stationary night blindness, cones Rod dystrophy, end-stage age-related macular degeneration, maculopathy, early-onset severe retinal dystrophy, color blindness, ocular albinism, oculocutaneous albinism, Stargardt disease, choroideremia, spinocerebellar ataxia type 7 (SCAT), mucopolysaccharides for use in the treatment of lysosomal storage diseases that affect the cornea, including MPS IV and MPS VII, retinoblastoma, intraocular melanoma, hypertensive retinopathy.

In certain embodiments, agents of the invention can be used for the treatment or prevention of diseases affecting the inner ear.

In certain embodiments, the agent is
a. intravitreal administration, particularly intravitreal injection, or b. Administered by subretinal administration.

In certain embodiments, the agent is delivered to the posterior segment, anterior segment, sclera, choroid, conjunctiva, iris, lens, or cornea.

Also for treating a condition selected from rod-cone dystrophy including retinitis pigmentosa, cone-rod dystrophy including macular degeneration and congenital stationary night blindness (CSBN1) in a patient in need thereof. comprising administering to a patient an AAV capsid and/or a viral vector comprising nucleic acid sequences according to the above description is within the scope of the present invention.

Also a dosage form for the prevention or treatment of a condition selected from cone-rod dystrophy, rod-cone dystrophy and congenital stationary night blindness, wherein the AAV capsid and/or according to one of the above aspects of the invention Alternatively, dosage forms are provided that include the nucleic acid sequences.

The agents and methods disclosed herein provide substantial benefits even at early disease stages with minimal damage to PR, and vectors driven by the present invention are more potent than any existing alternatives.

Whenever alternatives to a single separable feature, such as an AAV serotype protein or capsid peptide insert sequence or a medical indication, are described herein as an "embodiment", such It should be understood that the alternatives may be freely combined to form separate embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and benefits can be derived. These examples illustrate the invention but are not intended to limit its scope.

FIGURE LEGENDS Figure 1 Selected capsid variants compared to selected backbone AAV2 (Y252, 272, 444, 500, 700, 730F) hereinafter referred to as AAV2 (M6) and the state-of-the-art synthetic capsid AAV2 (7m8). is the cell specificity of expression for All of the AAVs were packaged as self-complementary (scAAV) expressing eGFP or mCitrine under the two ubiquitous promoters hEF1a and CMV to eliminate possible bias of one promoter towards certain retinal cell types. Expression in photoreceptors (ONL) is significantly enhanced for the novel mutant after intravitreal injection, in contrast to AAV2(M6) and AAV2(7m8). 2.5 μl injected intravitreally is equivalent in titer to 1E+11 vg/ml (excluding Cap14 at 3E+10). Mean±s.e.m. counted for 4 retinas d. . INL, inner nuclear layer; GCL, ganglion cell layer; DAPI, number of all cell bodies stained with nuclear stain DAPI.
FIG. 2. Strong pan-retina expression throughout the ONL for the indicated novel capsid mutants at 100× reduced functional titers compared to state-of-the-art AAV2 (7m8). All injections were titer equivalent and delivered 7.4E+7 vg into the vitreous of C57BL/6 mice. A. Low magnification image focusing on ONL of mouse retina wholemount after transduction with scCap5-CMV-EGFP. B-C. Vertical retinal cryosection. Micrographs reveal the potency of the novel capsid at lower titers compared to AAV2 (7m8).
Figure 3. Comparison of transduction after intravitreal injection of 2.5 μl scAAV into eyes of C57BL/6 mice (described, for example, in van Wyk et al., PLoS Biol, 2015.13(5): p.e1002143). is done). mCitrine expression under the control of the human Ef1a promoter, the titer is equivalent to 1E+11 vg/ml. Micrographs of vertical immunolabeled retinal cryosections showed that scAAV2 (Cap3) and scAAV2 (Cap5) penetrated well into the ILM and retinal layers, and either state-of-the-art AAV (7m8) or crude library backbone AAV2 (M6). It is clear that the expression efficiency is unique in ONL photoreceptors when compared with .

Example 1 Generation of Peptide Display Libraries The AAV capsid is icosahedral with 60 subunits. At the three-fold axis of the AAV capsid that is formed when the capsid subunits are assembled, a spike-like protrusion (peak) is formed that corresponds to the most exposed region of the capsid. In the AAV2 capsid, the highest peak is located at amino acid (AA) 453 and the second highest peak is located at AA 587/588 (AAV2-VP1 numbering). These peaks accept peptide insertions without interfering with viral capsid assembly and represent important sites for host interaction, receptor binding and immunogenicity. To generate a peptide display library, we additionally contained six tyrosine to phenylalanine mutations (Y252, 272, 444, 500, 700, 730F), designated AAV2 (M6). A randomly selected heptapeptide was inserted between _N587 and _R588 of the VP1 open reading frame of AAV2 (Table 2). YF and TV mutations (Buening & Srivastava, Mol Ther Methods Clin Dev. 2019 Jan 26;12:248-265.doi:10.1016/j.omtm.2019.01.008) reduce ubiquitination and thus reduce proteasomal degradation of viral particles once internalized by the cell (Petrs-Silva et al., Mol Ther, 2011.19: p.293-301.), thereby increasing transduction efficiency, It was previously shown that variants potentially favor the process of selection in intracellular evolution where the variants are very rare at early stages. Random insertions can be made at homologous sites in the GH loop (loop IV) of other capsid serotypes, as shown in Table 3. The approach to how the inventors developed the library incorporated that of Perabo and colleagues (Perabo et al., Mol Ther, 2003.8: 151-157). Two unique restriction sites, AscI and NotI, were introduced between amino acids 587 and 588 of the AAV2 (M6) genome (Table 2). The introduced DNA fragment forming AscI and NotI sites encoded a stop codon flanked by alanine linkers (AAAstopAA). Next, a randomly selected pool of single-stranded 7-mers with NNB codons was
5′-TTGGCGCGCCGCVNNVNNVNNVNNVNNVNNVNNGGCGGCCGCTTTTTTCCTGA-3′ (SEQ ID NO: 25)
(Eurofins Genomics) and converted to a pool of dsDNA fragments by second strand synthesis using the antisense primer 5'-CTCAAGGAAAAAGC-3' (SEQ ID NO: 26). To generate a 7mer display library, dsDNA random oligonucleotides were cloned into the AscI-NotI sites of the modified AAV (M6) genome, thereby replacing the stop codon. Viral libraries were generated such that each viral genome was packaged or wrapped within the capsid protein variants it encodes (genotype-phenotype coupling). In addition, all capsids were produced such that only one type of peptide was present in each of the 60 subunits. In this way, functional improvements identified through selection can be associated with genomic sequences encoding this improved function contained within the viral capsid.

Example 2 Selection of Peptide Mutants This library was subjected to positive selection in our laboratory-generated C57BL/6_Opto-mGluR6 mice that express the red fluorescent marker FP635 in retinal ON bipolar cells. (van Wyk et al., PLoS Biol, 2015.13(5): p.e1002143). This mouse line is known to have ON bipolar cells located deep within the retina and are not permissive for AAV transduction, resulting in favorable properties for transducing ON bipolar cells. The selection was made on the rationale that well-penetrating AAV and variants with .alpha. Briefly, as described in (van Wyk et al., PLoS Biol, 2015.13(5): p.e1002143; van Wyk et al., Front Neurosci, 2017.11: p.161), 5-8 transgenic mice, 4-6 weeks old, are injected intravitreally in both eyes with 2.5 μl of the iodixanol-purified library with a genome titer of approximately 5×10 ¹¹ viral genomes (vg)/ml. bottom.

After 10 days, the eyes were enucleated and the retinas were gently treated with papain protease and detached, followed by fluorescence-activated cell sorting (FACS) for the ON bipolar cell population. Successful virions were then PCR amplified from DNA extracts, further cloned and repackaged for subsequent injection steps.

At each cloning step of the 7mer oligos into the AAV2 (M6) genome, four ligations were performed in parallel and 12 clones from each pool were plated and sequenced to determine convergence of the library after each in vivo selection step. Decided. By this in vivo directed evolution process, positive selection was applied to create ON-type bipolar cell-permissive AAV mutants, similar to the natural evolution process.

From the in vivo selection library, 81,738 mutant capsid genes were identified by next-generation sequencing. The 15 heptapeptide sequences from the top 67 ranked were characterized by high overall transduction efficiencies (accumulated in NGS), preference for ON bipolar cell targeting and early selection stages, as outlined in Table 1. Or combinations of motifs that appeared promising with respect to their amino acid sequence (ie containing negatively charged or proline residues) were selected for functional determination and characterization. A key feature for all of the library variants is the separation of the positive charges (R ₅₈₅ and R ₅₈₈ ) of the heparan sulfate proteoglycan binding motif of AAV2 throughout the insertion. However, peptide sequences with the addition of two alanines in the linker sequence can regain the ability to bind heparan sulfate proteoglycans (Uhrig... Buening, Gene Therapy). Among the 7mer inserts, there was a moderate preference for certain positions, eg charged amino acids at positions 1 or 2 and/or positions 5 or 6 and polar amino acids such as Cys or Ala at position 7 (Table 1).

Example 3 Determination of Novel Capsid Mutants Recombinant forms of AAV2(M6)~7mer~ were cloned for all 15 selected peptides of Table 1. We packaged with the scCMV-EGFP transgene (CMV: cytomegalovirus promoter). Except for two mutants (Cap8 and Cap10), all capsids packaged properly with titers of 10 ¹¹ -10 ¹² vg/ml. Three weeks after intravitreal injection into adult mice, strong expression was observed for Cap3, Cap5, Cap7, Cap9, Cap11, Cap13 and Cap14, mainly in photoreceptors (Table 1, FIGS. 1-3), but also Some expression was observed in cells of the inner retinal layer such as bipolar cells, amacrine cells and Müller cells, and very little in retinal ganglion cells. A direct link with the productivity of AAV2 (7m8), which is also an evolved AAV2 variant (Dalkara et al., Sci Transl Med, 2013.5:p.189ra76.) and is considered to be currently state-of-the-art for intravitreal injection. In a relative comparison, our novel mutant penetrated the retina significantly better and transduced significantly more photoreceptor cells, but significantly fewer ganglion cells (Fig. 1, 1-3). The above results demonstrate that eight preselected mutants (Cap3, Cap5, Cap7, Cap9 Cap11, Cap13 and Cap14; Table 1) were further transformed into the schEf1a-EGFP-mCitrine transgene (hEf1a: human Ef1a promoter, "strong" natural ubiquitous mammalian promoters) and was confirmed by intravitreal injection of equivalent titers of 1×10 ¹¹ vg/ml into adult mice.

Similarly, the new mutants transduced significantly higher numbers of photoreceptors than controls AAV2 (M6) and AAV (7m8) of comparable titers. Consequently, selected mutants transduce photoreceptors in the outer nuclear layer (ONL) with superior internal limiting membrane and retinal penetration properties compared to AAV2(M6) and AAV2(7m8). (Fig. 1).

Cap14 packaged with scCMV-EGFP and schEf1a-EGFP and injected at 3E+10 vg/ml performed significantly better than AAV2(7m8) injected at the same low dose (FIGS. 1, 2), compared to typical in mice at 1E+12 vg/ml. It performed even better than AAV2(7m8) at the lowest doses commonly used. This is promising in terms of using lower doses in patients to reduce toxicity and immune response issues.

All 15 selected clones ranked among the top 67 out of 60,884 peptides sequenced from isolated ON bipolar cell fractions of C57BL/6_Opto-mGluR6 mice (van Wyk et al., PLoS Biol. 2015.13(5): p.e1002143).

The Cap gene of the library consists of VP3 (grey sequence), the tyrosine to phenylalanine (YF) mutation is underlined, and the base pair numbering refers to the entire VP1 sequence.

Claims (17)

An adeno-associated virus (AAV) capsid polypeptide that is at positions 453 or 587-592, particularly positions 587-592, more particularly position 587 of an AAV serotype 2 capsid or that of another serotype of AAV comprising a peptide insert at a homologous position, said peptide insert comprising:
SASEAST (Cap3; SEQ ID NO:10), DTRPHDQ (Cap5; SEQ ID NO:11), EHYNSTC (Cap7; SEQ ID NO:12), PNPNCTL (Cap9; SEQ ID NO:13), TPPSITA (Cap11; SEQ ID NO:14), CGESSYL (Cap12; number 15), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17)
in particular said insert is selected from SASEAST (Cap3; SEQ ID NO:10), TPPSITA (Cap11; SEQ ID NO:14), PRTPHTA (Cap13; SEQ ID NO:16) and ELCDGFA (Cap14; SEQ ID NO:17) adeno-associated virus (AAV) capsid polypeptide. An adeno-associated virus capsid polycomprising or consisting essentially of a sequence having at least (≧) 85% identity, particularly ≧90%, more particularly ≧95% identity to SEQ ID NO:001 a peptide,
characterized by an insert at position 587, 588, 589, 590, 591 or 592, particularly at position 587, 588 or 589, more particularly at position 587, and said insert comprises SEQ ID NO: 10, 11, 12 , 13, 14, 15, 16, 17. An adeno-associated virus capsid polypeptide which is or comprises a peptide sequence selected from any one of 13, 14, 15, 16, 17. 3. The adeno-associated virus capsid polypeptide of claim 2, characterized by at least 90% of the biological activity of a sequence selected from SEQ ID NO:2-SEQ ID NO:9. 4. The adeno-associated virus capsid polypeptide of claim 2 or 3, which is or comprises a sequence selected from SEQ ID NO:2-SEQ ID NO:9. 5. The adeno-associated virus capsid polypeptide of any one of claims 1-4, comprising or consisting essentially of an amino acid sequence selected from SEQ ID NO:2-SEQ ID NO:9. a. at positions 252, 272, 444, 500, 700, 704 and 730, specifically at positions 252, 272, 444, 500, 704 and 730, more specifically at positions 252, 272, 444, 500, 700 and 730 one or several tyrosine to phenylalanine substitutions, and/or b. one or several threonine to valine substitutions, particularly T491V
or a capsid other than an AAV2 capsid, and said capsid is characterized by a. and b. characterized by one or several tyrosine to phenylalanine substitutions and/or one or several threonine to valine substitutions in homologous positions of the capsid as described in claim 1 or the adeno-associated virus capsid polypeptide according to 2. A nucleic acid sequence encoding an AAV capsid polypeptide according to any one of claims 1-6. 8. A nucleic acid sequence according to claim 7, which is a self-complementary or single-stranded vector genome structure, in particular a self-complementary vector genome. 9. A nucleic acid sequence according to claim 7 or 8, comprising a transgene. 10. Nucleic acid sequence according to claim 9, wherein the transgene encodes a suitable protein, siRNA, shRNA or CRISPR/Cas-gRNA cassette sequence. 10. The nucleic acid sequence of claim 9, wherein said transgene encodes a light sensitive protein. A nucleic acid sequence according to any one of claims 9 to 11, wherein said transgene is under the control of a promoter sequence operable in mammalian cells, particularly retinal cells, more particularly human retinal cells. 13. The nucleic acid sequence of claim 12, wherein said promoter is a ubiquitous or cell-specific promoter. 13. The nucleic acid sequence of claim 12, wherein said promoter is selected from the CMV immediate early promoter or the hEf1a promoter. An agent selected from an AAV capsid polypeptide according to any one of claims 1 to 6 and a nucleic acid sequence according to any one of claims 7 to 14 for use in medicine/as a medicament. a. retinal cells or retinal pigment epithelial cells, and/or b. An AAV capsid polypeptide according to any one of claims 1 to 6 and any one of claims 7 to 14 for use in the treatment of a condition affecting photoreceptors, bipolar cells, ganglion cells or amacrine cells. An agent selected from the nucleic acid sequences described in Clause. from an AAV capsid polypeptide according to any one of claims 1-6 and a nucleic acid sequence according to any one of claims 7-14, for any of the uses according to claims 15 or 16 A selected agent comprising
a. intravitreal administration, particularly intravitreal injection, or b. Agents administered by subretinal administration.

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